1. Field of the Disclosure
The present disclosure relates to a method for obtaining human microglial precursor cells, comprising: (a) providing a cell population comprising neural precursor cells, wherein the cell population is obtainable from embryoid bodies differentiated from human pluripotent stem cells; (b) differentiating the cell population comprising neural precursor cells into microglial precursor cells by culturing in medium comprising a growth factor selected from the group consisting of insulin and insulin-like growth factors; (c) expanding and enriching microglial precursor cells in medium comprising a growth factor selected from the group consisting of insulin and insulin-like growth factors and 10 to 150 ng/ml GM-CSF; and (d) isolating microglial precursor cells comprising CD45-positive cells.
2. Discussion of the Background Art
In this specification, a number of documents including patent applications and manufacturer's manuals is cited. The disclosure of these documents, while not considered relevant for the patentability of this disclosure, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
Microglia are the resident immune cells of the central nervous system (CNS) and constitute about 10 to 20% of all glial cells in the adult CNS (Banati, 2003; Vaughan and Peters, 1974). The origin of microglia is still unclear. It was suggested that microglia appear in two waves, firstly in the neuroepithelium with unknown origin (Chan et al., 2007) and secondly in the brain during fetal development derived from the hematopoietic system and of mesodermal origin (Block and Hong, 2007; Chan et al., 2007).
Microglia respond to damage signals coming from injured tissue by undergoing activation of immune defence programs and proliferation (Ransohoff and Perry, 2009). Thus, microglia are responsible for the first line of the innate immune response in the CNS (Biber et al., 2007; Block et al., 2007; Hanisch and Kettenmann, 2007). Microglia are believed to remain in a resting stage under healthy physiological conditions. This stage is characterized by a ramified morphology and low expression of immunological molecules. In order to perform their surveillance function, microglia are highly dynamic during the resting stage and screen their environment. It is estimated that microglia can scan the entire brain parenchyma every few hours (Hanisch and Kettenmann, 2007; Nimmerjahn et al., 2005).
Under pathological conditions like injury or inflammation microglia become activated immune cells that show an amoeboid morphology, migrate to and within the lesion site, can clear apoptotic cells by phagocytosis and release a wide range of soluble factors that include neurotrophins and immunomodulatory factors (Biber et al., 2007; Block et al., 2007; Hanisch and Kettenmann, 2007). However, in some neurodegenerative diseases like Alzheimer's disease and multiple sclerosis, microglia become over-activated and have detrimental effects on neurons by releasing cytotoxic factors like nitric oxide and tumor necrosis factor-alpha (Block et al., 2007).
Microglial function is often studied using primary microglial cells, which are isolated and enriched from mixed glial cultures derived from the brains of postnatal mice or rats. A restricted number of microglial cells are obtained by a shaking procedure from mixed glial culture flasks (Giulian and Baker, 1986). Optionally, a purified population of microglial cells can be obtained using density gradients and flow cytometry sorting (Ford et al., 1995). Human primary microglia have also been obtained in very limited numbers from patients undergoing neurosurgery or from autopsy brains obtained after a short post mortem interval (Lafortune et al., 1996)
However, the obtained number of primary microglia is very limited in rodents and humans, which complicates classical biochemistry studies, systematic screening tests, or cell therapy approaches. In addition to primary cells, a murine microglial cell line (BV2) was developed by oncogenic transformation of primary microglia (Blasi et al., 1990; Bocchini et al., 1992). Furthermore, an immortalised human microglial cell line (HMO6) was developed through retroviral transduction of human embryonic telencephalon tissue with v-myc (U.S. Pat. No. 6,780,641). However, a drawback of all these cell lines is that they showed altered cytokine profile and changes in their migratory capacity (Horvath et al., 2008).
Recently, differentiation of microglia-like cells from mouse embryonic stem (ES) cells was described using a five-step protocol following neuronal differentiation (Tsuchiya et al., 2005). Tsuchiya et al. succeeded in differentiating Mac1+ cells into macrophages as well as into microglia and in isolating microglial cells that were positively stained for Iba1 and CD45, by a density gradient method. The isolated cells showed morphological characteristics of primary microglia and migrate from the bloodstream to brain parenchyma in mice. However, these cells were not described to survive and proliferate in culture (Tsuchiya et al., 2005). Recently, several microglial precursor cell lines were generated from murine ES cells (Napoli et al., 2009). The murine ES cell-derived microglial precursor (ESdM) lines were propagated in culture and expanded to high cell numbers. ESdM were indistinguishable by their cell surface receptors from primary microglia and showed migratory and phagocytic capacity comparable to primary microglia. After intracerebral transplantation in postnatal mice, they engrafted as microglial cells into the brain tissue.
However, despite the above described advances in the establishment of microglia precursor cell cultures, there is still the need to provide methods for the preparation of high quality human microglial precursor cells that can be obtained in large quantities.